Cardiovascular disease (CVD) is the leading cause of death in the world. One type of CVD, atherosclerosis, typically occurs in regions of disturbed blood flow, and its onset results in the stiffening of arteries and the creation of raised plaques which increase the chance of complete artery blockage. If blood supply to the brain is stopped as a result of this blockage, stroke occurs. The biophysical aspects associated with the mechanical effects of artery stiffening and how they relate to immune function will be investigated simultaneously in the proposed research plan. The main hypothesis of this proposal is that the mechanisms of transendothelial migration (TEM) by immune cells are affected by the organization and stiffness of the endothelial cell (EC) layer. Three specific aims are proposed to investigate this hypothesis. The first aim is to design and characterize an in vitro model of the vascular endothelium. The morphology, protein expression, stiffness, traction forces, F-actin distribution, and focal adhesions of the ECs will be examined as the underlying stiffness and shear stress are varied. The second aim is to investigate leukocyte migration along the EC monolayer as a function of underlying stiffness and shear stress. Leukocyte migration along the EC layer will be quantified using several parameters such as speed, random motility coefficient, and distribution of turning angles, and the traction forces exerted by the ECs will also be quantified as a measure of changes in force transmission through the ECs. The third aim is to investigate leukocyte TEM through ECs as a function of underlying stiffness and shear stress. The effects of leukocyte TEM on EC mechanotransmission will be investigated by examining the traction forces, F-actin distribution, and focal adhesions of the ECs. With the completion of the proposed research plan, we will more fully understand the role the ECs play as force transmitters during cellular TEM, and how this process might be altered in stiffened, atherosclerotic tissue. Stroke is a type of cardiovascular and neurological disease which often occurs due to blockage and stiffening of the arteries leading to the brain. This proposal investigates how stiffened, atherosclerotic tissue might affect immune cell migration into the diseased arteries, and how forces are transmitted through the vascular endothelium during this process. Ultimately, our goal is to find better ways to predict onset of a stroke before it occurs, so that treatment may be possible.